Combining a Genetically Engineered Oxidase with Hydrogen‐Bonded Organic Frameworks (HOFs) for Highly Efficient Biocomposites

Abstract Enzymes incorporated into hydrogen‐bonded organic frameworks (HOFs) via bottom‐up synthesis are promising biocomposites for applications in catalysis and sensing. Here, we explored synthetic incorporation of d‐amino acid oxidase (DAAO) with the metal‐free tetraamidine/tetracarboxylate‐based BioHOF‐1 in water. N‐terminal enzyme fusion with the positively charged module Zbasic2 strongly boosted the loading (2.5‐fold; ≈500 mg enzyme gmaterial −1) and the specific activity (6.5‐fold; 23 U mg−1). The DAAO@BioHOF‐1 composites showed superior activity with respect to every reported carrier for the same enzyme and excellent stability during catalyst recycling. Further, extension to other enzymes, including cytochrome P450 BM3 (used in the production of high‐value oxyfunctionalized compounds), points to the versatility of genetic engineering as a strategy for the preparation of biohybrid systems with unprecedented properties.

Purification of H-BM3, H-BlSP: H-BM3 and H-BlSP were purified with a HisTrap™ FF 5 mL column on a ÄKTA-system (ÄKTAprime plus or ÄKTA go, Cytiva, Vienna, Austria) with UV detection ( Figure S2-S3). Purification was at 4°C. Buffer A (50 mM HEPES, pH 7.5, 500mM NaCl, 20 mM imidazole) was used for the loading of the cell free extract. Buffer B (50 mM HEPES, pH 7.5, 500mM NaCl, 500 mM imidazole) was used for gradient elution. The flow rate was 3 ml min -1 . Typically, 20-30 mL cell-free extract (from ~5 g cell pellet) was loaded per purification Gradient elution was performed for 20 column volumes (100 mL). The purified enzyme was pooled and the buffer was exchanged by ultrafiltration (Vivaspin® Ultrafiltration Unit, Sartorius, Göttingen, Germany) to HEPES buffer (50 mM, pH 7.5). Purified enzymes were concentrated to a final concentration of 5-25 mg/mL and directly used for immobilization experiments or stored at -20°C. For H-HAD4 the purification did not achieve the desired amount of active enzyme, therefore it was not further examined in immobilization studies.

I6. Fourier transform infrared spectroscopy (FTIR).
FT-IR spectra were recorded on a Bruker ALPHA FTIR spectrometer (Bruker corporation, Billerica, MA, USA) using the ATR accessory with a diamond window in the range of ̃ 400 -4000 cm -1 , 256 scans, resolution 2 cm -1 . Samples were dropcast on the diamond window and dried using a nitrogen stream, prior to the measurement.

I8. Confocal laser scanning microscopy (CLSM).
Z-DAAO was tagged with Invitrogen Alexa Fluor™ 647 Protein Labelling Kit (Thermo Fisher Scientific, USA) according to the user guide for detection of the immobilized enzyme by CLSM.
Sample preparation. Briefly, the NHS ester (or succinimidyl ester) of Alexa Fluor® 647 was used to label primary amines (R-NH2) of Z-DAAO. 50 µL of sodium bicarbonate (1 M in ddH2O) was added to 500 µL of Z-DAAO (2 mg/mL). The reaction solution was transferred to a vial containing the reactive dye and a magnetic stirrer at incubated at 22°C for 1h. To remove unreacted dye a Zeba™ Dye and Biotin Removal Column (Thermo Fisher Scientific, USA) contained in the labelling kit was used. The column was washed with bicarbonate buffer (0.2 M, pH 9.4) three times, by centrifugation at 1000 rcf for 2 min, prior to applying the reaction solution, followed by centrifugation for 1000 rcf for 2 min. The flowthrough was collected and used for immobilization. After immobilization, 10 µL sample was dropped on a microscope slide, with a cover glass and sealed using clear nail-polish.
Image acquisition and processing. CLSM was performed by excitation with a 635 nm laser, using a Leica TCS SPE (Leica microsystems, Germany). The laser intensity was set to 100% and the gain set to 1200. The acquired images were cropped and recolored using Affinity Designer 1.10.4.1198 (Serif (Europe) Ltd., Nottingham, UK).
XRD patterns were acquired using a Rigaku® SmartLab II (Rigaku Europe SE, Neu-Isenburg, Germany) equipped with a Cu anode (9 kW, λ=1.5406Å). Prior the analysis the powder samples were dropcasted on a piece of Si (100) and dried overnight at 22°C.

I11. ζ-potential.
ζ-potential measurements via electrophoretic light scattering were performed using the litesizer 500 (Anton Paar® GmbH, Graz, Austria). Sample preparation was performed by dissolving each sample in filtered (0.2µm filter) ddH2O to reach a final Z-DAAO concentration of ~0.1 mg/mL for free and immobilized Z-DAAO. Material without Z-DAAO was diluted in an equivalent amount (1:10) after synthesis. Capillary of the zeta potential cuvette was filled with ~100 µL sample, capped and measured.

I12. Determination of protein concentration.
The protein concentration before and after immobilization was determined according to the Bradford assay [11] (ROTI®Quant, Carl Roth, Karlsruhe, Germany) or the bicinchoninic acid assay [12] (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Rockford, USA) using Albumin Fraction V (bovine serum) as a standard for calibration ( Figure S5-S6). For enzymes encapsulated in ZIF-8 and MAF-7 the Bradford assay showed no interference by MOF precursors. For Z-DAAO encapsulated in BioHOF-1, the BCA assay showed no interference by the HOF precursors. UV-VIS spectroscopy was performed on a FLUOstar ® Omega multi-mode microplate reader (BMG Labtech, Ortenberg, Germany).

I13. Enzymatic activity assays.
DAAO: In a standard experiment the enzymatic activity of DAAO and immobilized DAAO was determined by monitoring the initial O2 consumption rate during the conversion of D-methionine (Scheme S1). [13] Typically the linear initial-rate of O2 consumption after an initial equilibration period (~15s) and within the first 2 min of the reaction was used for linear regression analysis ( Figure S7). O2 concentrations were measured with a fiber-optic oxygen meter consisting of a FireStingO2 (FSO2-4) control unit with an OXROB10 probe (PyroScience GmbH, Aachen, Germany). The reaction set-up consisted of an open glass vial containing 4 mL reaction mix in a water bath (30°C) with magnetic stirring (6 x 3 mm, 400 rpm). The reaction mix (4 mL) consisted of D-methionine (20 mM) and DAAO or immobilized DAAO in air-saturated HEPES buffer (20 mM, pH 8). The specific activity of purified DAAO and Z-DAAO varied between 45-75 U mgenyzme -1 depending on expression batch and storage time. [14] The conversion of D-serine and cephalosporin c was performed with a final substrate concentration of 10 mM (Scheme S1). Scheme S1. Scheme of the reactions catalyzed by D-amino acid oxidase. Conversion of (a) d-methionine, (b) d-serine and (c) cephalosporin C BM3: The activity of BM3 and immobilized BM3 was measured through the initial O2 consumption rate during the hydroxylation of lauric acid at 30°C. [4] The reaction mix (4 mL) contained lauric acid (2 mM), NADP + (0.2 mM), glucose (200 mM), glucose dehydrogenase (1 mg/mL) and free or immobilized Z/H-BM3 in air-saturated HEPES buffer (50 mM, pH 7.4).

HAD4:
The activity of HAD4 and immobilized HAD4 was determined discontinuously by measuring the released phosphate during the conversion of α-glucose 1-phosphate. [6,15] The reaction was performed in a thermomixer at 30°C. The reaction mix (1 mL) consisted of MgCl2 (25 mM), NaCl (100 mM), α-glucose 1-phosphate (15 mM) and HAD4 or immobilized HAD4. Every 10 min 100 µL sample was taken and the reaction was stopped by adding 100 µL 1 M NaOH. After centrifugation for 2 min at 15000 rpm the amount of released phosphate was measured according to Saheki et al. [16] with modifications on a FLUOstar ® Omega multi-mode microplate reader (BMG Labtech, Ortenberg, Germany). Briefly, Molybdate reagent was prepared containing 15 mM ammonium molybdate and 100 mM Zinc acetate. The working reagent consisted of 4 parts Molybdate reagent and 1 part 10% ascorbic acid reagent (4+1). As a standard a 1 M KH2PO4 solution was prepared and diluted appropriately. 10 µL sample was mixed with 150 µL working reagent and incubated for 15 min at 25°C, subsequently, the absorbance was measured at 850 nm.

I14. Protease stability.
Free Z-DAAO and Z-DAAO@BioHOF-1 was incubated in a buffer solution containing 5 mg mL -1 trypsin from porcine pancreas at 37°C for 1 h. As a control, Z-DAAO and Z-DAAO@BioHOF-1 was incubated under the same conditions, excluding trypsin.

I15. Recycling of Z-DAAO@MAF-7 and Z-DAAO@BioHOF-1.
Recycling of each material was performed in 2 mL tubes on and end-over-end rotator at a reaction volume between 1 mL and 1.2 mL to ensure sufficient O2 supply. Each recycling step was performed for 2 min at 30°C. 20 mgwet weight mL -1 material was employed for each reaction. The reaction medium contained 20 mM HEPES (pH 8) and 10 mM D-methionine. After each cycle, the catalyst was recovered by centrifugation (20000 rcf for 2 min at 4°C) and reused in freshly prepared medium.

I16. Characterization of immobilization performance.
The protein yield ( ) was calculated as follows: Where is determined by measuring the protein concentration in the supernatant after immobilization.
Protein loading ( ) was calculated as follows: = Where is the mass of the immobilized biocatalyst and the mass of carrier.
The specific activity of the material ( ) was determined as follows: = Where is the activity of the immobilized biocatalyst and the mass of the carrier.
To determine the effectiveness of the immobilization the effectiveness factor (Ƞ) was calculated:

Ƞ =
Where is the specific activity of the enzyme per mass of enzyme bound to the carrier (U mgenzyme -1 ).
Apparent Michaelis-Menten kinetic parameters were assessed by measuring the O2 consumption rate at varying D-methionine concentrations (0.1 -10 mM). Nonlinear regression analysis was performed using the Michaelis-Menten model: Where 0 is the initial rate of reaction, the maximum rate, the substrate concentration at 2 and [ ] the initial substrate concentration.

= * [ ]
Where is the turnover number and [ ] the enzyme concentration.

Phase optimization of Z-DAAO@ZIF-8 and Z-DAAO@MAF-7.
Initially, the immobilization of DAAO@ZIF-8 was explored using an established protocol [9] with a metal to ligand ratio (M/L) of 1:16 mM during the synthesis. To confirm the topology of each material the crystallinity of each biocomposite was monitored using powder x-ray diffraction (PXRD). PXRD patterns revealed that pure ZIF-8 formed with sod phase, whereas Z-DAAO@ZIF-8 leads to the formation of ZIF-L ( Figure S10). ZIF-L is a 2D ZIF with a decreased pore size (vs. ZIF-8). [17] To obtain porous ZIF-8 with sod topology, the synthesis conditions were optimized. The enzyme preparation contains 250 mM NaCl to minimize unspecific ionic interactions of Z-DAAO and its purification tag (Zbasic2). [2,5] We examined the addition of NaCl (50/100 mM) during the synthesis of ZIF-8, which lead to the formation of ZIF-L ( Figure S12). By increasing the concentration of 2-methyl-1H-imidazole (M/L = 1:32), ZIF-8 and Z-DAAO@ZIF-8 resulted in the formation of sod, in the presence of 100 mM NaCl (Figure S14). Therefore the M/L of 1:32 was chosen for the one-pot immobilization of Z-DAAO@ZIF-8. Next, the synthesis of Z-DAAO@MAF-7 was examined using PXRD. Initial one-pot immobilization of Z-DAAO@MAF-7 with a previously reported method [9] resulted in the formation of amorphous material ( Figure S11). In contrast to Z-DAAO@ZIF-8, NaCl did not affect the synthesis of MAF-7 ( Figure S13). However, to obtain crystalline Z-DAAO@MAF-7 the concentration of base (NH3(aq)), required in the synthesis of MAF-7, had to be increased (from 0.25% to >0.5% Figure S15). Although Z-DAAO influences the crystallinity of ZIF-8 and MAF-7 no such effect could be observed for Z-DAAO@BioHOF-1 (Figure 3a). The optimized immobilization protocols can be seen in I3 -I5. Interestingly, while Z-DAAO@ZIF-L/ZIF-8 was inactive, the amorphous MAF-7 lead to an increase in immobilization performance over Z-DAAO@MAF-7 with a sodalite topology ( Figure S38).                      Figure S26. Particle size analysis of ZIF-8 with inset particle distribution density plot. Where ±…represents the standard deviation. Image analysis was performed with FIJI. [10] Figure S27. Particle size analysis of Z-DAAO@ZIF-8 with inset particle distribution density plot. Where ±…represents the standard deviation. Image analysis was performed with FIJI. [10] Figure S28. Particle size analysis of MAF-7 with inset particle distribution density plot. Where ±…represents the standard deviation. Image analysis was performed with FIJI. [10] Figure S29. Particle size analysis of Z-DAAO@MAF-7 with inset particle distribution density plot. Where ±…represents the standard deviation. Image analysis was performed with FIJI. [10] Figure S30. Particle size analysis of BioHOF-1 with inset particle distribution density plot. Where ±…represents the standard deviation. Image analysis was performed with FIJI. [10] Figure S31. Particle size analysis of Z-DAAO@BioHOF-1 with inset particle distribution density plot. Where ±…represents the standard deviation. Image analysis was performed with FIJI. [10] Figure S32. Comparison of the particle size distribution. (a) particle diameter of ZIF-8 and Z-DAAO@ZIF-8, (b) particle diameter of MAF-7 and Z-DAAO@MAF-7, (c) particle length of BioHOF-1 and Z-DAAO@BioHOF-1, and (d) particle width of BioHOF-1 and Z-DAAO@BioHOF-1.            Figure S49. Oxygen time courses of Z-DAAO@BioHOF-1 (df=10) at different D-methionine concentrations (0.1 -10 mM) and fitted curves to determine the consumption rate. Linear regression after an initial equilibration period (see I10 DAAO Activity assay).      Residual activity / % Sample Figure S55. Oxygen time courses of Z-DAAO@MAF-7 after recycling. The reaction was performed with 20 mgwet weight mL -1 biocomposite, 10 mM D-methionine and 20 mM HEPES (pH 8) at 30°C after each the biocomposite was separated by centrifugation and reused in a fresh reaction mixture. Linear regression after an initial equilibration period (see I10 DAAO Activity assay).      [a] Theoretical specific activity of each material, calculated from the specific activity of each enzyme obtained in this work (Z-BM3 = 0.4 U mg -1 , Z-BlSP = 38.1 U mg -1 , Z-HAD4 = 0.8 U mg -1 ) and the reported protein loading of each material. Theoretical specific activities of each material were calculated assuming an ideal effectiveness factor (ƞ = 1).